Nucleosome structure has profound implications for DNA-templated processes and modulation of this structure is of critical importance for replication and transcription. Nucleosome structure must be loosened (reorganized) to facilitate polymerase passage and normal structure must be re-established in the wake of the elongating polymerase. Also, nucleosomes must be translated along DNA (remodeled) to allow access to promoter sequences and replication origins. Our goal is to understand the molecular basis for these activities and other important regulatory interactions. Our proposal is organized into three specific Aims, each of which focuses on a different protein or complex: Spt6 (Aim 1) promotes reformation of normal nucleosome structure following passage of RNA Pol II. It also makes a myriad of functionally important interactions and provides a link between the machineries for RNA transcription and processing. yFACT (Spt16-Pob3) (Aim 2) is a reorganizing complex that restructures nucleosomes to facilitate transcription initiation and passage of elongating RNA Pol II. It also performs an important (presumably equivalent) role in DNA replication. RSC (Aim 3) is an ATP driven remodeling complex that functions in the expression of numerous genes by mediating nucleosomal translocation. Our approach is highly collaborative and broad. Because our goal is to achieve detailed molecular understanding we will determine relevant structures by x-ray crystallography (Hill, PI). Because we aim to understand processes in a biological context we are exploiting the strengths of yeast genetics (Formosa, co-I; and Cairns, collaborator). The link between structural and in vivo studies is provided by biochemical analysis, which is ongoing in all three labs. Further strength is added by collaboration with an expert in electron microscopy (Andres Leschziner). We have made considerable progress toward the specific aims. Important structures have been determined and informative biochemical and genetic assays developed. Preliminary data indicate that considerable productivity will ensue and also lay the foundation for longer-term goals that carry more risk but also offer potential for high reward PUBLIC HEALTH RELEVANCE: The proteins/complexes Spt6, yFACT, and RSC mediate nucleosome dynamics and other events that regulate gene expression and DNA replication. We aim to advance understanding of function by a combination of determining relevant three-dimensional structures, biochemistry, and genetic analysis.